Assuntos
Regiões 3' não Traduzidas/genética , Análise Mutacional de DNA/métodos , Testes Genéticos/métodos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Mutação Puntual , Reação em Cadeia da Polimerase/métodos , Polimorfismo Genético , Protrombina/genética , Trombofilia/genética , Alelos , Automação , Análise Mutacional de DNA/instrumentação , Testes Genéticos/instrumentação , Humanos , Metilenotetra-Hidrofolato Redutase (NADPH2) , Reação em Cadeia da Polimerase/instrumentação , Reprodutibilidade dos Testes , Fatores de Risco , Sensibilidade e Especificidade , Taq Polimerase , Trombofilia/diagnóstico , Trombofilia/epidemiologia , Fatores de TempoRESUMO
The G1691A (Leiden) mutation of the factor V gene is the most prevalent identified cause of venous thrombosis. Therefore, we developed a new genetic test using the TaqMan system. With this assay which combines PCR amplification and detection reaction in one closed tube, a cohort of 234 patients with a history of thrombosis was screened. In parallel, amplification products of the same patients were screened with a previously described test using endonuclease digestion of PCR products followed by gel electrophoresis. Identical results were obtained by both methods. Among cases, 122 (52%) individuals were homozygous normal, 99 (42%) were heterozygous affected and 13 (5.5%) showed homozygous pattern for the Factor V Leiden mutation. Thus, it could be demonstrated that the new TaqMan assay is a robust, rapid and automated method for high throughput application which avoids time consuming and difficult post-PCR steps.